CN113981064B - Biomarker for detecting diabetic retinopathy, detection kit and application - Google Patents

Biomarker for detecting diabetic retinopathy, detection kit and application Download PDF

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CN113981064B
CN113981064B CN202111222357.0A CN202111222357A CN113981064B CN 113981064 B CN113981064 B CN 113981064B CN 202111222357 A CN202111222357 A CN 202111222357A CN 113981064 B CN113981064 B CN 113981064B
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diabetic retinopathy
mirna
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CN113981064A (en
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蒋沁
石泽惠
姚牧笛
韩笑言
李秀苗
刘畅
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Eye Hospital Nanjing Medical University
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Abstract

The invention belongs to the technical field of biomedical detection, and discloses a biomarker for detecting diabetic retinopathy, a detection kit and application thereof, wherein the biomarker for detecting diabetic retinopathy is miRNA-375-3p, and the nucleotide sequence of the marker is shown as SEQ ID NO:1 is shown. The invention determines that the change of the expression level of miRNA-375-3p has obvious correlation with the occurrence of diabetic retinopathy. miRNA-375-3p can be used as a biomarker for early diagnosis of diabetic retinopathy, and the progression of the diabetic retinopathy and relapse conditions after treatment are periodically evaluated. The invention provides an important reference basis for early diagnosis, prognosis judgment and early intervention treatment of the diabetic retinopathy, has higher actual clinical value, can perform intervention and treatment as soon as possible, and reduces unnecessary treatment and cost of non-high-risk relapsed patients.

Description

Biomarker for detecting diabetic retinopathy, detection kit and application
Technical Field
The invention belongs to the technical field of biomedical detection, and particularly relates to a biomarker for detecting diabetic retinopathy, a detection kit and application.
Background
Currently, diabetic retinopathy is the major blinding eye disease, and retinal microvascular disease is considered to be the major cause. The main manifestations are that after the blood-retina barrier is damaged by hyperglycemia, microvascular occlusion is caused, so that neovascularization is caused, the visual function is reduced, and the life quality of patients can be seriously affected. In the early stages of diabetic retinopathy, the patient may not have obvious symptoms until significant vision loss or visual field loss begins, and the associated ischemic ocular disease is not detected, but the visual impairment is irreversible. At present, the treatment modes aiming at the diabetic retinopathy mainly comprise medicines aiming at primary diseases, intravitreal medicine injection, retinal laser, operation and the like, and the symptomatic treatment modes can hardly reverse the optic nerve degeneration and the visual deterioration. Therefore, the early diagnosis and prognosis evaluation of the occurrence of the diabetic retinopathy are urgently needed to find out a novel biomarker with small trauma, strong operability and high sensitivity.
Micrornas (mirnas) are a class of endogenous single-stranded non-coding RNAs of about 22nt in length that also remain highly conserved during evolution. The miRNA is combined with a 3' end non-coding region of target mRNA to block the translation process or directly cause mRNA degradation, so that the transcription level and the post-transcription level are regulated and controlled, and the miRNA is involved in various biological processes, is closely related to tumors, cardiovascular diseases, metabolic system diseases and the like, and is expected to become a novel disease diagnosis biomarker.
However, there is no report of miRNA-375-3p as a biological marker for diabetic retinopathy diagnosis. Therefore, a new biomarker for detecting diabetic retinopathy is needed to fill the blank of the prior art.
Through the above analysis, the problems and defects of the prior art are as follows: the early diagnosis and prognosis evaluation of the diabetic retinopathy lack biomarkers with small traumatism, strong operability and high sensitivity, and the miRNA-375-3p is not reported as a biological marker for the diagnosis of the diabetic retinopathy at present.
The difficulty in solving the above problems and defects is: the screening of the novel biomarker needs to be based on a sequencing technology, qPCR technology verification, cell phenotype experiment verification of the influence of marker intervention on cell functions and verification of the expression amount of a large amount of clinical samples. The validation process requires scientific rigorous experimental design and meaningful statistical analysis.
The significance of solving the problems and the defects is as follows: a novel disease diagnosis marker is provided for early diagnosis and prognosis evaluation of diabetic retinopathy patients, and a new idea is provided for diagnosis and treatment of diabetic retinopathy.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a biomarker for detecting diabetic retinopathy, a detection kit and application thereof, and aims to solve part of the problems in the prior art or at least alleviate part of the problems in the prior art.
The invention is realized in such a way that the diabetic retinopathy detection biomarker is miRNA-375-3p, and the nucleotide sequence of the marker is shown in SEQ ID NO:1 is shown.
The invention also provides a primer for detecting the biomarker for detecting the diabetic retinopathy, wherein the sequence of the primer is shown as SEQ ID NO:2, respectively.
The invention also aims to provide a real-time fluorescence quantitative PCR detection kit for detecting the diabetic retinopathy by applying the biomarker for detecting the diabetic retinopathy, which comprises a PCR amplification system, wherein the PCR amplification system comprises 2 xEZ-Probe qPCR Master Mix for microRNA, probe 10 mu M, a specific qRT-PCR upstream primer, namely a specific recognition miR-375-3p or U6, and a universal 3' end downstream primer.
Further, the miRNA-375-3p specific qRT-PCR upstream primer sequence is shown in SEQ ID NO:2, the sequence of the U6 quantitative PCR upstream primer is shown as SEQ ID NO:3, respectively.
Further, the real-time fluorescence quantitative PCR detection kit for diabetic retinopathy further comprises a reverse transcription reaction system, wherein the reverse transcription reaction system comprises gDNA Remover,4 xmiRNA RT Buffer, miRNA RT Enzyme Mix, and nucleic free ddH 2 O。
Further, the real-time fluorescence quantitative PCR detection kit for diabetic retinopathy further comprises an RNA extraction system, wherein the RNA extraction system comprises Trizol reagent, chloroform, absolute ethyl alcohol and DEPC ddH 2 O，ddH 2 O, isopropanol.
The invention also aims to provide a screening method for detecting biomarkers by using the diabetic retinopathy, which comprises the following steps:
step one, sample preparation: constructing a rat diabetic retinopathy DR cell model, and taking endothelial progenitor cells BM-EPC from rat bone marrow of an experimental group and a control group;
step two, screening differential expression miRNA: analyzing miRNA related to the occurrence of diabetic retinopathy by adopting an Illumina sequencing platform;
step three, after the experimental result of sequencing analysis is verified by adopting quantitative PCR, a target point is finally determined for subsequent determination by combining GO and KEGG signal path analysis;
and step four, verifying the expression difference of the target miRNA-375-3p in the aqueous humor and the plasma of patients with diabetic retinopathy and cataract patients.
Further, in the first step, the experimental group is a high sugar stimulation group, and n =3; the control group is a Ctrl group, n =3; RNA was extracted using TRIzol reagent and stored at-80 ℃ until use.
In the second step, the analyzing of the miRNA associated with the occurrence of diabetic retinopathy includes:
after the Sample is qualified, constructing a library by using a small RNA Sample Pre Kit; the method comprises the steps of utilizing special structures of the 3 'end and the 5' end of small RNA (the 5 'end has a complete phosphate group, and the 3' end has a hydroxyl group), taking total RNA as an initial sample, adding linkers at the two ends of the small RNA, carrying out reverse transcription to synthesize cDNA, and then carrying out PCR amplification and PAGE gel electrophoresis separation to obtain a cDNA library. After the library is constructed, firstly carrying out primary quantification by using the qubit2.0, then carrying out Q-PCR by using a Bio-RAD CFX 96 fluorescent quantitative PCR instrument and a Bio-RAD KIT iQ SYBR GRN, and accurately quantifying the effective concentration of the library to ensure the quality of the library; generating a Cluster on a cBot by using a TruSeq PE Cluster Kit v3-cBot-HS (Illumina) reagent, and then running a sequencing program on an Illumina sequencing platform to obtain sequencing reads; analyzing small RNA sequencing data of an Illumina platform, analyzing the expression condition of small RNAs in species by comparing with a reference genome, and carrying out differential expression analysis.
The invention also aims to provide the application of the diabetic retinopathy detection biomarker in preparing a diabetic retinopathy diagnostic reagent, in particular to the application in preparing a reagent for detecting the diabetic retinopathy based on a real-time fluorescent quantitative PCR technology.
The invention also aims to provide application of the antisense nucleotide of the diabetic retinopathy detection biomarker in preparing a medicament for treating diabetic retinopathy, wherein the sequence of the antisense nucleotide is shown as SEQ ID NO:4, respectively.
The invention also aims to provide application of the diabetic retinopathy detection biomarker in preparation of a reagent for prognosis evaluation after treatment of diabetic retinopathy, in particular to application in preparation of a reagent for prognosis evaluation after treatment of diabetic retinopathy based on a real-time fluorescence quantitative PCR technology.
By combining all the technical schemes, the invention has the advantages and positive effects that: the biomarker for detecting the diabetic retinopathy provided by the invention can be used for diagnosing the human diabetic retinopathy and judging the disease development according to the expression level of the miRNA-375-3p of an individual. The miRNA-375-3p antisense nucleotide is complementary with a target miRNA-375-3p, inhibits or blocks the conversion and expression of genes, or induces RNase H to recognize or cut the miRNA-375-3p to lose the function, so that the miRNA-375-3p antisense nucleotide can be used as a medicine for treating eye diseases related to diabetic retinopathy. Therefore, the invention provides a new application of miRNA-375-3p, which can be used as a diagnostic reagent for early diagnosis of diabetic retinopathy.
Through high-throughput sequencing screening and probe quantitative PCR verification, the invention discovers that the miRNA-375-3p has obvious correlation with the occurrence of diabetic retinopathy. The invention utilizes the probe method real-time fluorescence quantitative PCR technology, and by detecting the obvious difference of the miRNA-375-3p expression quantity in the aqueous humor and the plasma of a diabetic retinopathy patient relative to a cataract patient, the miRNA-375-3p can be used for screening clinical diabetic retinopathy patients, and theoretical basis is provided for early intervention of diabetic retinopathy.
The invention determines that the change of the expression level of miRNA-375-3p has obvious correlation with the occurrence of diabetic retinopathy. By interfering the expression of miRNA-375-3p in endothelial progenitor cells, the change of the expression level of miRNA-375-3p is verified to significantly influence the cell function in functional experiments of cell activity, proliferation, migration and tube formation. After the miRNA-375-3p is expressed and silenced in endothelial progenitor cells, the activity of the cells can be obviously enhanced, the proliferation and migration of the cells are promoted, and the apoptosis of the cells is inhibited. The miRNA-375-3p can be used as a biomarker for early diagnosis of diabetic retinopathy, and the progression of the diabetic retinopathy and relapse conditions after treatment are periodically evaluated.
The invention provides an important reference basis for early diagnosis, prognosis judgment and early intervention treatment of the diabetic retinopathy disease, has a larger practical clinical value, can be used for screening high risk groups and recurrent groups of the diabetic retinopathy to intervene and treat as soon as possible, and reduces unnecessary treatment and medical cost of non-high risk recurrent patients.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram of screening miRNA related to the occurrence of diabetic retinopathy by MicroRNA high-throughput sequencing analysis provided by the embodiment of the invention.
FIG. 1A is a qualitative schematic of boxplot analytical sequencing, in which each 6 samples were mixed to form a biological replicate, eliminating individual variation, according to an embodiment of the present invention.
Fig. 1B is a schematic diagram of a scattergram showing the miRNA expression difference between Diabetic Retinopathy (DR) and non-diabetic retinopathy (Ctrl) samples as a whole according to an embodiment of the present invention.
Fig. 1C is a schematic diagram of a clustering map provided in the present invention for analyzing miRNA expression differences between diabetic retinopathy and non-diabetic retinopathy samples from an overall perspective.
FIG. 2 is a schematic diagram of quantitative PCR analysis of difference of miR-375-3p expression in the aqueous humor of experimental group (diabetic retinopathy) and control group (cataract) patients according to the embodiment of the invention.
FIG. 3 is a schematic diagram of the quantitative PCR analysis of the expression change of miR-375-3p in the plasma of experimental group (diabetic retinopathy) and control group (cataract) patients.
FIG. 4 is a schematic diagram for analyzing the influence of miR-375-3p intervention on the pathological process related to rat Diabetic Retinopathy (DR) endothelial cells, provided by the embodiment of the invention.
FIG. 4A is a schematic diagram of quantitative PCR analysis provided in the embodiment of the invention for analyzing the effect of miR-375-3p mimics transfection bone marrow derived endothelial progenitor cells (BM-EPC) on miR-375-3p expression.
FIG. 4B is a schematic diagram of the effect of analyzing miR-375-3p intervention on BM-EPC activity by MTT colorimetry provided by the embodiment of the invention.
FIG. 4C is a schematic diagram of analyzing the effect of miR-375-3p intervention on BM-EPC proliferation by using a CCK-8 kit provided by the embodiment of the invention.
FIG. 4D is a schematic diagram of analyzing the effect of miR-375-3p intervention on BM-EPC apoptosis by Calcein-AM and Propidium Iodide (PI) immunofluorescence staining, wherein an upper graph is a fluorescence staining graph, and a lower graph G is a statistical analysis graph.
FIG. 4E is a schematic diagram of analyzing the effect of miR-375-3p intervention on BM-EPC migration function by using a Transwell cell migration experiment provided by the embodiment of the invention, wherein an upper graph is a cell migration staining graph, and a lower graph H is a statistical analysis graph.
FIG. 4F is a schematic diagram of analysis of the effect of miR-375-3p intervention on the angiogenesis activity of BM-EPC by a Matrigel gel angiogenesis assay provided in the embodiments of the present invention, in which the upper panel is a cell tube formation diagram, and the lower panel I is a statistical analysis diagram.
FIG. 5 is a schematic diagram of the quantitative PCR detection results of serum samples before and after the operation treatment of diabetic retinopathy patients, wherein the abscissa represents the serum samples before and after the operation treatment, and the ordinate represents the miRNA-375-3p expression level.
Fig. 6 is a flowchart of a screening method according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a biomarker for detecting diabetic retinopathy, a detection kit and application thereof, and the invention is described in detail with reference to the accompanying drawings.
The biomarker for detecting the diabetic retinopathy provided by the embodiment of the invention is miRNA-375-3p, and the nucleotide sequence of the marker is shown as SEQ ID NO:1 is shown.
As shown in fig. 6, the screening method provided in the embodiment of the present invention includes the following steps:
s101, sample preparation: constructing a rat diabetic retinopathy DR cell model, and taking an endothelial progenitor cell BM-EPC from rat bone marrow of an experimental group and a control group;
s102, screening of differential expression miRNA: analyzing miRNA related to the occurrence of diabetic retinopathy by adopting an Illumina NovaSeq 6000 sequencing platform;
s103, verifying the experimental result of sequencing analysis by adopting quantitative PCR, and finally determining a target point for subsequent determination by combining GO and KEGG signal path analysis;
s104, verifying the expression difference of the target miRNA-375-3p in the aqueous humor and the plasma of patients with diabetic retinopathy and cataract.
The normal temperature in the following embodiments of the present invention refers to a natural room temperature condition in four seasons, and is not subjected to additional cooling or heating treatment, and is generally controlled at a normal temperature of 10-30 ℃, preferably 15-25 ℃.
The technical solution of the present invention is further described below with reference to specific examples.
Example 1
The invention is realized in such a way that the biomarker for detecting the diabetic retinal lesion based on the real-time fluorescence quantitative PCR technology is miRNA-375-3p, and the nucleotide sequence is shown as SEQ ID NO. 1. Wherein, SEQ ID NO.1: miR-375-3p nucleotide sequence: r no-miR-375-3p MIMAT0005307: UUUGUUCGUUCGGCUCGCGUGA.
The detection primer of the marker is shown as SEQ ID NO. 2. Wherein, SEQ ID NO is 2: miR-375-3p-F: TTTGTTCGTTCGGCTCG.
A Chinese medicinal composition for treating diabetesThe real-time fluorescence quantitative PCR detection kit for retinopathy comprises a PCR amplification system, wherein the PCR amplification system comprises 2 xEZ-probe qPCR Master Mix for microRNA (including Ex Taq enzyme, dNTP Mix, mg) 2+ Tli RNaseH, TB Green) 100 μ L; probe,1 tube, 1. Mu.l; miRNA-375-3p specific qRT-PCR upstream primer, 1 tube, 10 mu M,100 mu L/tube; universal 3' end downstream primer, 1 tube, 10 muM, 100 muL/tube; u6 quantitative PCR forward primer, 1 tube, 10. Mu.M, 100. Mu.L/tube.
Furthermore, the miRNA-375-3p specific qRT-PCR upstream primer is shown as SEQ ID NO.2, and the U6 quantitative PCR upstream primer sequence is shown as SEQ ID NO. 3. Wherein, SEQ ID NO:3: CCTGCTTCGGCAGCACA.
Further, the kit also comprises a reverse transcription reaction system, wherein the reverse transcription reaction system comprises a gDNA remover (comprising DNase and buffer), 1 tube and 55 ul/tube; miRNA RT Enzyme Mix, tube 1, 110 ul/tube; 4 × miRNA RT Buffer (including Oligo dT-Universal tag universal reverse transcription primers, dNTPs and Buffer), 1 tube, 275 ul/tube; nuclear free ddH 2 O,1 ml/tube.
The method specifically comprises the following steps: gDNA Remover,4 XmiRNA RT Buffer, miRNA RT Enzyme Mix, nucleic acid free ddH 2 O。
Further, the kit also comprises an RNA extraction system, wherein the RNA extraction system comprises a Trizol reagent,1 tube and 2000 mu L/tube; chloroform, 1 tube, 500 μ L/tube; absolute ethyl alcohol, 1 tube, 8000 mul/tube; DEPC ddH 2 O,1 tube, 1000. Mu.L/tube; ddH2O,1 tube, 2000. Mu.L/tube; isopropanol, 8000 μ L/tube.
The biomarker is applied to the preparation of a diagnostic reagent for diabetic retinopathy, in particular to the application of the biomarker in the preparation of a diagnostic reagent for detecting diabetic retinopathy based on a real-time fluorescent quantitative PCR technology.
The antisense nucleotide of the biomarker is applied to the preparation of the medicine for treating diabetic retinopathy.
The biomarker is applied to the preparation of a prognosis evaluation reagent after the treatment of diabetic retinopathy, in particular to the application of the biomarker in the preparation of the prognosis evaluation reagent after the treatment of diabetic retinopathy based on a real-time fluorescence quantitative PCR technology.
In the embodiment of the invention, the miRNA-375-3p is determined to be used as the biomarker for diagnosing the diabetic retinopathy, namely the screening method comprises the following steps:
the first step is as follows: sample preparation: a rat Diabetic Retinopathy (DR) cell model was constructed, and rat bone marrow-derived endothelial progenitor cells (BM-EPC) were taken from the experimental group (high glucose stimulated group, n = 3) and the control group (Ctrl, n = 3). RNA was extracted using TRIzol (Invitrogen) reagent and stored at-80 ℃ until use.
The second step is that: screening of differential expression miRNA: and analyzing miRNA related to the occurrence of diabetic retinopathy by adopting an Illumina sequencing platform. The analysis comprises the following specific steps: after the Sample is detected to be qualified, constructing a library by using a small RNA Sample Pre Kit, taking total RNA as an initial Sample by utilizing the special structures of the 3 'end and the 5' end of the small RNA (the 5 'end has a complete phosphate group, and the 3' end has a hydroxyl group), adding a joint at the two ends of the small RNA, carrying out reverse transcription to synthesize cDNA, and then carrying out PCR amplification and PAGE gel electrophoresis separation to obtain a cDNA library. After the library is constructed, firstly, using Qubit2.0 to carry out preliminary quantification, then using a Bio-RAD CFX 96 fluorescent quantitative PCR instrument and a Bio-RAD KIT iQ SYBR GRN to carry out Q-PCR, and accurately quantifying the effective concentration of the library to ensure the quality of the library; generating a Cluster on the cBot by using a TruSeq PE Cluster Kit v3-cBot-HS (Illumina) reagent, and then running a sequencing program (SE 50) on an Illumina sequencing platform to obtain sequencing reads; analyzing small RNA sequencing data of an Illumina platform, analyzing the expression condition of small RNAs in species by comparing with a reference genome, and carrying out differential expression analysis.
The third step: and (3) verifying the experimental result of sequencing analysis by adopting quantitative PCR, and then combining GO and KEGG signal path analysis to finally determine a target point for subsequent determination.
The fourth step: verifying the expression difference of the target miRNA-375-3p in the aqueous humor and the plasma of patients with diabetic retinopathy and cataract patients.
The miRNA-375-3p antisense nucleotide is complementary with a target miRNA-375-3p, inhibits or blocks the conversion and expression of genes, or induces RNase H to recognize or cut the miRNA-375-3p to lose functions, so the miRNA-375-3p antisense nucleotide can be used as a medicament for treating eye diseases related to diabetic retinopathy. The sequence of the antisense nucleotide is shown as SEQ ID NO:4: ucacgcgagccgaacgaacaaa.
Example 2: verification of correlation of miRNA-375-3p and diabetic retinopathy
MicroRNA high-throughput sequencing analysis screens and verifies miRNA related to diabetic retinopathy.
The first step is as follows: sample preparation: a rat Diabetic Retinopathy (DR) cell model was constructed, and rat bone marrow-derived endothelial progenitor cells (BM-EPC) were taken from the experimental group (high glucose stimulated group, n = 3) and the control group (Ctrl, n = 3). RNA was extracted using TRIzol (Invitrogen) reagent and stored at-80 ℃ until use.
The second step is that: screening of differential expression miRNA: and analyzing miRNA related to the occurrence of diabetic retinopathy by adopting an Illumina sequencing platform. The analysis comprises the following specific steps: after the Sample is detected to be qualified, constructing a library by using a small RNA Sample Pre Kit, taking total RNA as an initial Sample by utilizing the special structures of the 3 'end and the 5' end of the small RNA (the 5 'end has a complete phosphate group, and the 3' end has a hydroxyl group), adding a joint at the two ends of the small RNA, carrying out reverse transcription to synthesize cDNA, and then carrying out PCR amplification and PAGE gel electrophoresis separation to obtain a cDNA library. After the library is constructed, firstly, using Qubit2.0 to carry out preliminary quantification, then using a Bio-RAD CFX 96 fluorescent quantitative PCR instrument and a Bio-RAD KIT iQ SYBR GRN to carry out Q-PCR, and accurately quantifying the effective concentration of the library to ensure the quality of the library; generating a Cluster on the cBot by using a TruSeq PE Cluster Kit v3-cBot-HS (Illumina) reagent, and then running a sequencing program (SE 50) on an Illumina sequencing platform to obtain sequencing reads; the distribution of each sample is shown in FIG. 1A. Small RNA sequencing data for the Illumina platform were analyzed for small RNA expression in species by alignment with a reference genome and differential expression analysis was performed (see figure 1B). The distribution of differentially expressed mirnas for each sample is shown in figure 1C.
The third step: the experimental results of the high-throughput sequencing analysis were verified by quantitative PCR (see table 1); and finally determining a target point, namely miR-375-3p (hsa-miR-375-3 p) for subsequent determination by combining GO and KEGG signal path analysis. The nucleotide sequence of miR-375-3p is shown in SEQ ID NO. 1.
TABLE 1 Experimental results of quantitative PCR validation of high throughput sequencing analysis
Figure BDA0003313077850000111
The fourth step: the expression difference of the target miR-375-3p in the aqueous humor of the experimental group (diabetic retinopathy) and the control group (cataract) patients is verified (as shown in figure 2, and figure 2 shows 50 typical cases which are respectively selected from 200 cases in each group).
Example 3: expression detection of miR-375-3p in plasma
The first step is as follows: plasma sample separation
Blood samples of 200 cases of each patient were collected from the experimental group (diabetic retinopathy) and the control group (cataract), and plasma was separated therefrom by density gradient centrifugation using a heparin anticoagulation tube for miRNA detection. The centrifugation conditions were 4 ℃ at 12000rpm, and 10min.
The second step is that: RNA extraction from plasma
TRIzol is added to the separated plasma sample, and the mixture is allowed to stand at room temperature for 10min to allow the sample to be sufficiently lysed (note: the sample can be stored at-70 ℃ for a long period of time without further processing). Adding 200 μ l chloroform into 1ml TRIzol, vigorously shaking, mixing, standing at room temperature for 3-5min, and naturally separating phases. Centrifuge at 12000rpm for 15min at 4 ℃. The sample will be divided into three layers: yellow organic phase, intermediate layer and colorless aqueous phase, RNA is mainly in the aqueous phase, the aqueous phase is transferred to a new tube. An equal volume of ice-cold isopropanol was added to the supernatant and left at room temperature for 15min. Centrifugation was carried out at 12000rpm for 10min at 4 ℃ and the supernatant was discarded, and RNA was precipitated at the bottom of the tube. To the RNA pellet, 1ml of 75% ethanol (prepared with RNase-free water) was added, and the pellet was suspended by gently shaking the centrifuge tube. 1ml of 75% ethanol was added per 1ml of TRIzol. Centrifuging at 8000rpm at 4 deg.C for 5min, and discarding the supernatant. After air-drying at room temperature, 50. Mu.l of RNase-free water was added to the precipitate to dissolve RNA sufficiently, and the mixture was stored at-70 ℃.
The third step: RNA quality detection
Measuring the concentration of RNA at the absorbance positions of 260nm and 280nm by adopting an ultraviolet spectrophotometer; the ratio of A260/A280 of the RNA solution is the RNA purity, and the ratio ranges from 1.8 to 2.1. Meanwhile, the quality of RNA is detected by combining agarose gel electrophoresis, and the RNA is observed and photographed under ultraviolet transmitted light.
The fourth step: reverse transcription of RNA to obtain cDNA sample
Reverse Transcription Using the microRNA Reverse Transcription Kit from EZB, the following were added to the reaction system (20. Mu.l) provided in the Kit:
Figure BDA0003313077850000121
the above system was placed in Rnase-free 0.2 μ l EP tubes and inverted to cDNA according to the following procedure: the cDNA was stored at-20 ℃ for 15min at 37 ℃,10min at 42 ℃ and 3min at 95 ℃.
The fifth step: probe method quantitative PCR
The PCR reaction system (50. Mu.l) was quantified by probe method using EZ-probe qPCR Master Mix for microRNA (ROX 2 plus) from EZB, and the following was prepared according to the system provided in the specification: 2 xEZ-Probe qPCR Master Mix for microRNA 25. Mu.l, probe (10. Mu.M) 1. Mu.l, upstream primer 2. Mu.l of specific qRT-PCR (specific recognition miR-375-3p or U6), downstream primer 2. Mu.l, sample cDNA 5. Mu.l to be detected, RNase free ddH 2 Make up to 50. Mu.l of O.
U6 quantitative PCR upstream primer 5'-CCTGCTTCGGCAGCACA-3';
miR-375-3p quantitative PCR upstream primer 5'-TTTGTTCGTTCGGCTCG-3';
PCR conditions were as follows: 95 ℃ for 5min,92 ℃ for 10s; 30s at 60 ℃;40 cycles.
And a sixth step: data analysis
Respectively carrying out real-time fluorescence quantitative PCR detection on target RNA and internal reference RNA on the same sample, carrying out normalization treatment on the target RNA by taking the expression quantity of the internal reference as a reference, then analyzing the relative expression quantity of the target RNA by adopting a Delta Ct method commonly used in the field, subtracting the Ct value of the target gene miR-375-3p of all samples from the Ct value of the internal reference gene U6 of the sample to obtain the Delta Ct value (Delta Ct) of all samples, and expressing the formula as Delta Ct = Ct (target gene miR-375-3 p) -Ct (internal reference gene U6). Finally, the disease susceptibility of the patient to be tested is judged by comparing the expression difference of miR-375-3p of the sample to be tested and the target sample (for example, 50 representative cases selected from 200 cases in each group are shown in figure 3).
The seventh step: interpretation of results
The expression level of the target miR-375-3p is detected by a probe method quantitative PCR method, and the delta Ct range of each group of control group samples is obtained, which is shown in Table 2. And comparing the delta Ct of the samples to be detected, and analyzing the difference between the experimental group and the control group. Further analyzing whether the value of each sample is in the range of a control group, and if the delta Ct of the sample to be detected is in the range of the delta Ct of the control group or is smaller than the range of the delta Ct, determining that the sample to be detected is negative for diabetic retinopathy; if the Δ Ct is greater than this Δ Ct range, it is considered to be positive for diabetic retinopathy.
TABLE 2 Delta Ct ranges for each group of samples
mean Ct-U6 Ct-mean of microRNA-375-3p
Aqueous humor of control group patients 15.2-23.7
Control group patient plasma 14.3-24.2
Test group patients aqueous humor 2.6-3.9
Plasma of patients in the experimental group 1.4-2.5
According to the results, 200 diabetic retinopathy and 200 non-diabetic retinopathy patients diagnosed by clinical imaging diagnosis are respectively collected for miR-375-3p serum content analysis. The accuracy of the detection based on the method of miR-375-3p is evaluated, and the results in Table 3 show that the accuracy of a serum sample is 82.5%, the sensitivity is 84.0%, and the miR-375-3p can be used as a biological marker for diagnosing the diabetic retinopathy.
TABLE 3 results of diagnosis of diabetic retinopathy with miR-375-3p as biomarker
Figure BDA0003313077850000141
Eighth step: based on in vitro experiments, the miR-375-3p is disclosed to be involved in the regulation of the diabetic retinopathy process by regulating the function of EPC cells from the aspect of basic principle. In vitro experiments, mimics (double strands) with the sequence 5'-UUUGUUCGUUCGGCUCGCGUGA-3' and the antisense strand 5'-ACGCGAGCCGAACGAACAAAUU-3' and miR-375-3p inhibitor with the sequence 5'-UCACGCGAGCCGAACGAACAAA-3' were used to transfect EPC to interfere miR-375-3p expression. Analyzing the influence of miR-375-3p transfected EPC on miR-375-3p expression by using quantitative PCR (quantitative PCR), and analyzing the influence of miR-375-3p mimics transfected bone marrow-derived endothelial progenitor cells (BM-EPC) on miRNA-375-3p expression by using the quantitative PCR shown in figure 4A; the influence of miR-375-3p intervention on the activity of BM-EPC is analyzed by adopting an MTT colorimetric method as shown in figure 4B; the effect of miR-375-3p intervention on the proliferation of BM-EPC is analyzed by using a CCK-8 kit as shown in FIG. 4C; FIG. 4D shows the effect of miR-375-3p intervention on BM-EPC apoptosis analyzed by Calcein-AM and Propidium Iodide (PI) immunofluorescence staining, wherein the upper graph is a fluorescence staining graph, and the lower graph 4G is a statistical analysis graph; FIG. 4E shows a diagram of analyzing the effect of miR-375-3p intervention on BM-EPC migration function by using a Transwell cell migration experiment, in which the upper graph is a cell migration staining graph, and the lower graph 4H is a statistical analysis graph; FIG. 4F is a graph of the effect of miR-375-3p intervention on the angiogenic activity of BM-EPC using a Matrigel gel angiogenesis assay, wherein the upper panel is a cell tubulation graph and the lower panel 4I is a statistical analysis graph; the result shows that miR-375-3p silencing in-vitro experiments has a protective effect on BM-EPC injury induced by high glucose hypochondriac stress; overexpression of miR-375-3p can aggravate BM-EPC injury effect induced by high sugar stress; the miR-375-3p is suggested to regulate and control the function of BM-EPC cells, and further regulate and control the diabetic retinopathy process.
Example 4: feasibility application of miR-375-3p as prognosis evaluation marker
A first step; obtaining a blood sample to be examined
Serum was collected from 200 patients with diabetic retinopathy before and after treatment.
The second step: extraction of RNA from blood sample to be examined
a) Taking 0.25ml of serum sample or frozen serum, transferring the serum sample or frozen serum sample into a centrifuge tube, adding 1ml of Trizol, and repeatedly sucking up and down by using a liquid transfer gun until the cells are completely lysed;
b) Adding chloroform (1/5 volume of the sample solution and Trizol Reagent), covering the centrifugal tube cover tightly, oscillating vigorously for 15sec, and standing at room temperature for 5min;
c) Centrifuging at 4 ℃,12000g multiplied by 15min, taking out the centrifuge tube from the centrifuge, and dividing the homogenate into three layers at the moment: colorless supernatant, intermediate white protein layer and colored lower organic phase. The supernatant is sucked and transferred to another new centrifuge tube (the white middle layer is not sucked out);
d) Adding isopropanol with the volume of 1 time into the supernatant, and fully and uniformly mixing by vortex;
e) Centrifuging at 4 ℃ for 12000g multiplied by 10min, wherein after centrifugation, sediment appears at the bottom of a test tube, and discarding supernatant;
f) Adding 1ml 75% ethanol, slightly inverting by hand, centrifuging at 12000g for 5min, and discarding supernatant;
g) Adding 1ml of absolute ethyl alcohol, slightly inverting by hand, centrifuging at 12000g for 5min, and discarding the supernatant;
h) Drying at room temperature, adding appropriate amount of DEPC H 2 Dissolving O (promoting dissolution at 65 deg.C for 10-15 min), and gently blowing and beating the precipitate with a pipette if necessary;
i) The BioDrop uv-vis spectrophotometer measures the purity and concentration of RNA.
The third step: the RNA obtained was Reverse transcribed into cDNA and prepared using microRNA Reverse Transcription Kit from EZB according to the system (20. Mu.l) provided in the Kit as follows: gDNA Remover treated RNA 5. Mu.l, 4X miRNA RT Buffer 5. Mu.l, miRNA RT Enzyme Mix 2. Mu.l, nucleic free ddH 2 O8. Mu.l, inverted to cDNA according to the following procedure: the cDNA was stored at-20 ℃ for 15min at 37 ℃,10min at 42 ℃ and 3min at 95 ℃.
The fourth step: probe method quantitative PCR
The PCR reaction system (50. Mu.l) was quantified by probe method using EZ-probe qPCR Master Mix for microRNA (ROX 2 plus) from EZB, and the following was prepared according to the system provided in the specification: 2x EZ-Probe qPCR Master Mix for microRNA 25. Mu.l, probe (10. Mu.M) 1. Mu.l, upstream primer 2. Mu.l of specific qRT-PCR (specific recognition miR-375-3p or U6), downstream primer 2. Mu.l, sample cDNA 5. Mu.l to be tested, RNase free ddH 2 Make up to 50. Mu.l of O.
PCR conditions were as follows: 95 ℃ for 5min,92 ℃ for 10s; 30s at 60 ℃;40 cycles.
The fifth step: data analysis
The gene expression value is calculated by a Delta Ct method, and the amplification efficiency of the target gene and the reference gene is assumed to be close to 100% and the relative deviation is not more than 1Ct; Δ Ct = mean Ct for gene of interest-mean Ct for reference gene, where U6 is selected for reference gene.
And a sixth step: result judgment
And (4) calculating the delta Ct of the sample to be detected, and analyzing the change condition of the sample before and after the operation treatment. The results are shown in FIG. 5: collecting serum from diabetic retinopathy patients before and after treatment; extracting total RNA by using Trizol reagent, and obtaining cDNA of the total RNA by a reverse transcription PCR method; the expression level of the target miR-375-3p is detected by a probe method quantitative PCR method, and 50 representative cases selected from 200 cases are shown in figure 5. The expression difference of miR-375-3p in the serum of diabetic retinopathy patients before and after treatment is analyzed by probe quantitative PCR, and the result shows that the expression of miR-375-3p in the serum of diabetic retinopathy patients after treatment is obviously reduced, which indicates that miR-375-3p is expected to be a target for evaluating the curative effect and prognosis of the operation.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made by those skilled in the art within the technical scope of the present invention disclosed in the present invention should be covered within the scope of the present invention.
Sequence listing
<110> ophthalmic hospital of Nanjing medical university
<120> diabetic retinopathy detection biomarker, detection kit and application
<160> 4
<170> SIPOSequenceListing 1.0
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<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
uuuguucguu cggcucgcgu ga 22
<210> 2
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tttgttcgtt cggctcg 17
<210> 3
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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cctgcttcgg cagcaca 17
<210> 4
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<212> RNA
<213> Artificial Sequence (Artificial Sequence)
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ucacgcgagc cgaacgaaca aa 22

Claims (1)

  1. Application of antisense nucleotide of miRNA-375-3p in preparing reagent for enhancing activity of endothelial progenitor cells of patients with diabetic retinopathy, promoting proliferation and migration of endothelial progenitor cells of patients with diabetic retinopathy or inhibiting apoptosis of endothelial progenitor cells of patients with diabetic retinopathy, wherein the sequence of the antisense nucleotide is shown as SEQ ID NO:4, respectively.
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